Monolayer-forming islet cell culture from neonatal pig pancreas: Using sequential treatment with EDTA-dispase and monoiodoacetic acid for preparation and purification.

Abstract

A new method for the preparation and purification of a monolayer-forming islet cell culture from the neonatal pig pancreas is described. Single cell preparations of the pig pancreas were obtained by gently stirring the chopped pancreases in a medium containing sequential EDTA-dipase. Exposure of the cells to monoiodoacetic acid (10 micron) and BSA-gradient resulted in improved preparation, highly enriched in the endocrine cells. The cells were maintained in tissue culture medium 199 containing 5.5 mM D-glucose, with or without 0.1 mM 3-isobutyl-1-methylxanthine, or 16.7 mM D-glucose and 20% fetal calf serum. During a 6-day culture period, many small cell aggregates appeared in the media. The cell clusters attached to the bottom of the dish and formed monolayers. Provocative stimulation and radioimmunoassay for insulin showed the existence of numerous and viable B cells in the cell clusters. The other three types of islet cells were also demonstrated immunohistochemically in the cell cluster. It is concluded that this improved culture technique provides a useful tool for morphological and biochemical studies of islet cells and a potential source of material for transplantation of B-cells from the pancreas.